Highly Efficient and Enzymatic Regioselective Undecylenoylation of Gastrodin in 2-Methyltetrahydrofuran-Containing Systems

Highly efficient and regioselective acylation of pharmacologically interesting gastrodin with vinyl undecylenic acid has been firstly performed through an enzymatic approach. The highest catalytic activity and regioselectivity towards the acylation of 7′-hydroxyl of gastrodin was obtained with Pseudomonas cepacia lipase. In addition, it was observed the lipase displayed higher activity in the eco-friendly solvent 2-methyltetrahydrofuran-containing systems than in other organic solvents. In the co-solvent mixture of tetrahydrofuran and 2-methyltetrahydrofuran (3/1, v/v), the reaction rate was 60.6 mM/h, substrate conversion exceeded 99%, and 7′-regioselectivity was 93%. It was also interesting that the lipase-catalyzed acylation couldn’t be influenced by the benzylic alcohol in gastrodin. However, pseudomonas cepacia lipase displayed different regioselectivity towards gastrodin and arbutin.     

Immobilized Pseudomonas sp. lipase: A powerful biocatalyst for asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate

Pseudomonas sp. lipase was immobilized onto glutaraldehyde-activated Florisil^® support via Schiff base formation and stabilized by reducing Schiff base with sodium cyanoborohydride. The immobilization performance was evaluated in terms of bound protein per gram of support (%) and recovered activity (%). A 4-factor and 3-level Box–Behnken design was applied for the acylation of (±)-2-(propylamino)-1-phenylethanol, a model substrate, with vinyl acetate and the asymmetric acylations of other (±)-2-amino-1-phenylethanols with different alkyl substituents onto nitrogen atom such as (±)-2-(methylamino)-1-phenylethanol, (±)-2-(ethylamino)-1-phenylethanol, (±)-2-(butylamino)-1-phenylethanol and (±)-2-(hexylamino)-1-phenylethanol were performed under the optimized conditions. The optimal conditions were bulk water content of 1.8%, reaction temperature of 51.5 °C, initial molar ratio of vinyl acetate to amino alcohol of 1.92, and immobilized lipase loading of 47 mg mL^?1. (R)-enantiomers of tested amino alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an increase in the acylation yield and ee values of (S)-enantiomers. Enantiomeric ratio values were >200 for all the reactions. Our results demonstrate that the immobilized lipase is a promising biocatalyst for the preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation of (±)-2-amino-1-phenylethanols with vinyl acetate.     

Efficient regioselective acylation of andrographolide catalyzed by immobilized Burkholderia cepacia lipase

For the first time, PSL-C, an immobilized lipase from Burkholderia cepacia, was successfully applied to the regioselective acylation of andrographolide by vinyl acetate in acetone. FT-IR spectra demonstrated the occurrence of acylation reaction. The ¹³C NMR, ESI-MS and elemental analysis confirmed that the 14-acetylandrographolide was formed exclusively. Water activity and reaction temperature had a significant effect on the initial rate and the substrate conversion, but little effect on the regioselectivity of the reaction. The optimal water activity and reaction temperature were 0.11 and 50 °C, respectively. Under these conditions, the initial rate and substrate conversion were 50.2 mM h⁻¹ and 99.0%, respectively, after a reaction time of around 4 h. Besides, immobilized lipase also displayed higher operational stability and 83.5% of its original activity was maintained after being reused for eight batches.     

Production,partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis

Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24 h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The K_m and V_max values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 μmoles/mg min. The metal ions Ca²⁺, Mg²⁺ and Mn²⁺ had stimulatory effect on lipase activity, whereas Cu²⁺, Fe²⁺ and Zn²⁺ strongly inhibited the lipase activity. EDTA and PMSF at 10 mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.     

Genetical and biochemical evidence that the hydroxylation pattern of the anthocyanin B-ring in Silene dioica is determined at the p-coumaroyl-coenzyme a stage

In petals of Silene dioica, gene P controls the 3′-hydroxylation of the anthocyanin B-ring and the hydroxylation pattern of the hydroxycinnamoyl acyl group bound to the 4″'-hydroxyl group of rhamnose of anthocyanidin 3-rhamnosyl(1→6)glucoside-5-glucoside. In this paper, experiments are presented which show that gene P is involved in the hydroxylation of p-coumaroyl-CoA to caffeoyl-CoA, which is then used both as a precursor in anthocyanin biosynthesis and as a substrate for the final acylation.     

Janus-faced Enzymes Yeast Tgl3p and Tgl5p Catalyze Lipase and Acyltransferase Reactions

In the yeast, mobilization of triacylglycerols (TAGs) is facilitated by the three TAG lipases Tgl3p, Tgl4p, and Tgl5p. Motif search analysis, however, indicated that Tgl3p and Tgl5p do not only contain the TAG lipase motif GXSXG but also an H-(X)₄-D acyltransferase motif. Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both enzymes not only exhibited lipase activity but also catalyzed acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two enzymatic activities act independently of each other. We also showed that Tgl3p is important for efficient sporulation of yeast cells, but rather through its acyltransferase than lipase activity. In summary, our results demonstrate that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes.     

Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach

Lipases are widely used for a variety of biotechnological applications. Screening these industrial enzymes directly from environmental microorganisms is a more efficient and practical approach than conventional cultivation-dependent methods. Combined with activity-based functional screening, six clones with lipase activity were detected and a gene (termed lipZ01) isolated from a target clone with the highest lipase activity was cloned from an oil-contaminated soil-derived metagenomic library and then sequenced. Gene lipZ01 was expressed in Pichia pastoris GS115 and the molecular weight of the recombinant lipase LipZ01 was estimated by electrophoresis analysis to be approximately 50 kDa. The maximum activity of the purified lipase was 42 U/mL, and the optimum reaction temperature and pH value were 45 °C and 8.0, respectively. The enzyme was highly stable in the temperature range 35–60 °C and under alkaline conditions (pH 7–10). The presence of Ca²⁺ and Mn²⁺ ions could significantly enhance the activity of the lipase. The purified lipase preferentially hydrolysed triacylglycerols with acyl chain lengths ≥8 carbon atoms, and the conversion degree of biodiesel production was nearly 92% in a transesterification reaction using olive oil and methanol. Some attractive properties suggested that the recombinant lipase may be valuable in industrial applications.     

Biocatalytic synthesis,structural elucidation,antioxidant capacity and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin

Our present investigation describes the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids. The biocatalytic synthesis was optimized to achieve 81–98% yields, using immobilized lipase B, from Candida antarctica (Novozym 435®), in acetone at 45 °C. The synthesized esters have been analyzed by ¹H NMR, ¹³C NMR spectroscopy and evaluated for their antioxidant capacity and tyrosinase inhibition, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the greatest potential for inhibition of tyrosinase activity (p ?0.05) was exhibited by the α-linolenic acid ester of isoquercitrin.     

Synthesis of (−)-lobeline via enzymatic desymmetrization of lobelanidine

The bioactive alkaloid (?)-lobeline was synthesized via the stereoselective acylation (desymmetrization) of meso-lobelanidine by vinyl acetate in the presence of Candida antarctica lipase B.     

Enzymatic acylation of polar dipeptides: Influence of reaction media and molecular environment of functional groups

The enzymatic acylation of polar dipeptides was investigated. First, the Novozym435^®-catalyzed acylation of Lys-Ser, HCl exhibiting three potential acylable sites was carried out in organic media (2-methyl-2-butanol, oleic acid) and in an ionic liquid ([Bmim]⁺[PF₆]^?). In these reactions, the chemo-selectivity of the acylation was exclusively in favour of the N?-lysine acylation and the efficiency (substrate conversion) was demonstrated to be under control of the peptide solubility. The use of [Bmim]⁺[PF₆]^? permitted to significantly improve the dipeptide solubility, and to enhance both substrates conversion and initial rates of acylation reaction. In the three reaction media used, the O-acylated derivative of the dipeptide was never detected suggesting a weak reactivity of the serine hydroxyl group due to its molecular environment and particularly to the presence of a free carboxylic group known for its electroattractor property.Last, the acylation of a natural dipeptide (carnosine), exhibiting a very low solubility in organic solvents, was also performed. Carnosine was successfully N-acylated in 2-methyl-2-butanol, and a yield of 39% was obtained when improving the substrate solubility: a better dispersibility was obtained by application of a high pressure on the reaction medium just before starting the reaction.     

Cytokinin metabolism and the modulation of cytokinin activity in radish

The metabolism of zeatin, N⁶-(Δ²-isopentenyl) adenine, dihydrozeatin, zeatin-O-glucoside and dihydrozeatin-O-glucoside has been studied using derooted radish seedlings. The metabolites were identified by UV and GC/MS. The patterns of metabolism are compared and provide evidence that the O-glucosyl conjugates may be storage forms of the cytokinins.     

Lipase-catalyzed solid-phase synthesis of sugar esters. Influence of immobilization on productivity and stability of the enzyme

The lipase-catalyzed synthesis of 6-O-glucose palmitate in a mainly solid-phase system was investigated. Lipase from Candida antarctica B was immobilized on various carriers by physical absorption or covalent binding. Highest conversion (84%, 24 h) and productivity (0.69 mmol product per gram lipase and hour) were achieved with lipase immobilized on polypropylene (EP 100), whereas other carriers gave at maximum 46% conversion. A good agreement between aquaphilicity of the carrier and conversion was found. The lipase was reused six times and conversion decreased by only approximately 25%. The influence of temperature, organic solvent and fatty acid chain length on lipase stability was also investigated. For the latter, a correlation between the log P of the fatty acid (determined from hydrophobic fragment constants) and lipase stability was observed. Palmitic acid and the corresponding vinyl ester gave highest conversion in the acylation of β-d(+)-glucose, whereas tripalmitin, palmitic acid anhydride and the methyl ester allowed only low conversion. Beside β-d(+)-glucose, also other monosaccharides and trehalose were acylated.     

An extracellular solvent stable alkaline lipase from Pseudomonas sp. DMVR46: Partial purification,characterization and application in non-aqueous environment

The biosynthesis of esters is currently of much commercial interest because of the increasing popularity and demand for natural products among consumers. Biotransformation and enzymatic methods of ester synthesis are more effective when performed in non-aqueous media. In present study, an organic solvent stable Pseudomonas sp. DMVR46 lipase was partially purified by acetone precipitation and ion exchange chromatography with 28.95-fold purification. The molecular mass of the lipase was found to be ∼32 kDa. The partially purified lipase was optimally active at 37 °C and pH 8.5. The enzyme showed greater stability toward organic solvents such as isooctane, cyclohexane and n-hexane retaining more than 70% of its initial activity. The metal ions such as Ca²⁺, Ba²⁺ and Mg²⁺ had stimulatory effects on lipase activity, whereas Co²⁺ and Zn²⁺ strongly inhibited the activity. Also lipase exhibited variable specificity/hydrolytic activity toward different 4-nitrophenyl esters. DMVR46 lipase was further immobilized into AOT-based organogels used for the synthesis of flavor ester pentyl valerate in presence of organic solvents. The organogels showed repeated use of enzyme with meager loss of activity even upto 10 cycles. The solvent-stable lipase DMVR46 thus proved to be an efficient catalyst showing an attractive potency for application in biocatalysis under non-aqueous environment.     

Mass spectral studies of phytosterolins and a ketone from Trianthema pentandra

Mass spectral data of a phytosterolin and its tetraacetate from Trianthema pentandra show that sitosterol-d(+)-glucoside reported earlier¹ was a mixture of sitosteroI-d(+)-glucoside and stigmasterol-d(+)-glucoside. The structure of a ketone has been suggested as nonacos-1-en-4-one, on the basis of physicochemical data. The positions of the double bond and the carbonyl group have been established by MS and NMR data. This is the first report in plants of a C₂₉ ketone with a terminal double bond and carbonyl group at position 4.     

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.     

Highly regioselective enzymatic synthesis of 5′-O-stearate of 1-β-D-arabinofuranosylcytosine in binary organic solvent mixtures

In this paper, highly regioselective enzymatic acylations of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5′-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS–ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50°C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol·L⁻¹h⁻¹, 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13(¹³C) NMR and confirmed to be 5′-O-stearate of ara-C.     

Purification and Characterization of a DNA Topoisomerase I from Bifidobacterium breve

The monoacylation of (η⁶-1,2-benzenedimethanol)tricarbonylchromium (2) by vinyl acetate, palmitate and benzoate, alcoholysis of the corresponding diesters of 2 in n-butanol, and acylation of (η⁶-benzyl alcohol) tricarbonylchromium by (±)-vinyl 2-phenoxypropanoate and 2-phenylpropanoate were accomplished with lipase P (from P. fluorescens) and lipase CC (from C. cylindracea) to give optically active organometallic esters. Their configurations indicated that the stereoselectivity of each of these two lipases was in marked contrast. An active site model for them is proposed.     

Anaerobic Degradation of Flavonoids by Clostridium orbiscindens

An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as Clostridium orbiscindens by comparative 16S rRNA gene sequence analysis. The organism was tested for its ability to transform several flavonoids. The isolated C. orbiscindens strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively. Genistein and daidzein were not utilized. The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2′-glucoside were not cleaved. Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed. To investigate the numerical importance of C. orbiscindens in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity. Application of the probe to fecal samples from 10 human subjects proved the presence of C. orbiscindens in 8 out of the 10 samples tested. The numbers ranged from 1.87 × 10⁸ to 2.50 × 10⁹ cells g of fecal dry mass⁻¹, corresponding to a mean count of 4.40 × 10⁸ cells g of dry feces⁻¹.     

Novel and Highly Efficient Regioselective Route to Helicid Esters by Lipozyme TLL

Highly regioselective acylation of helicid with fatty acid vinyl esters catalyzed by the lipase from Thermomyces lanuginosus has been successfully performed for the first time. For the enzymatic caproylation of helicid, under the optimal conditions, initial reaction rate was 33.2 mM/h, and substrate conversion and regioselectivity were greater than 99%. In addition, the acyl recognition of the enzyme in the regioselective acylation of helicid was investigated. The results showed that although 6’-O-acyl derivatives of helicid were exclusively obtained with all the tested acyl donors, the enzymatic reaction rate varied widely with different acyl donors, presumably owing to their different interactions with the active site of the lipase. It is also interesting that the different configuration of only one hydroxyl group at C-3 in helicid couldn’t affect the lipase-catalyzed esterification and helicid has the same regioselectivity as that of D-glucose and arbutin.